Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A-related kinase 5

BACKGROUND Tubulins,building blocks of microtubules,are modified substrates of diverse post-translational modifications including phosphorylation,polyglycylation and polyglutamylation.Polyglutamylation of microtubules,catalyzed by enzymes from the tubulin tyrosine ligase-like(TTLL)family,can regulate interactions with molecular motors and other proteins.Due to the diversity and functional importance of microtubule modifications,strict control of the TTLL enzymes has been suggested.AIM To characterize the interaction between never in mitosis gene A-related kinase 5(NEK5)and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODS The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5(a.a.260–708)as bait and confirmed by immunoprecipitation.The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTS Here,we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity.We further show that NEK5 can also interact with TTLL5 and TTLL7.The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4.The same effects were observed after the expression of the catalytically inactive form of NEK5.This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSION Our results demonstrate,for the first time,the regulation of TTLL activity through phosphorylation,pointing to NEK5 as a potential effector kinase.We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

Posttranslational modifications of α-tubulin in alzheimer disease

Background:In Alzheimer disease(AD),hyperphosphorylation of tau proteins results in microtubule destabilization and cytoskeletal abnormalities.Our prior ultra-morphometric studies documented a clear reduction in microtubules in pyramidal neurons in AD compared to controls,however,this reduction did not coincide with the presence of paired helical filaments.The latter suggests the presence of compensatory mechanism(s)that stabilize microtubule dynamics despite the loss of tau binding and stabilization.Microtubules are composed of tubulin dimers which are subject to posttranslational modifications that affect the stability and function of microtubules.Methods:In this study,we performed a detailed analysis on changes in the posttranslational modifications in tubulin in postmortem human brain tissues from AD patients and age-matched controls by immunoblot and immunocytochemistry.Results:Consistent with our previous study,we found decreased levels ofα-tubulin in AD brain.Levels of tubulin with various posttranslational modifications such as polyglutamylation,tyrosination,and detyrosination were also proportionally reduced in AD brain,but,interestingly,there was an increase in the proportion of the acetylatedα-tubulin in the remainingα-tubulin.Tubulin distribution was changed from predominantly in the processes to be more accumulated in the cell body.The number of processes containing polyglutamylated tubulin was well preserved in AD neurons.While there was a cell autonomous detrimental effect of NFTs on tubulin,this is likely a gradual and slow process,and there was no selective loss of acetylated or polyglutamylated tubulin in NFT-bearing neurons.Conclusions:Overall,we suggest that the specific changes in tubulin modification in AD brain likely represent a compensatory response.